Current gene therapy techniques face critical challenges to translation including targeting incorrect cells, silencing of genes over time, delivery of large genes, manufacturing cost, and risk of permanently altering a patient?s germline DNA. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system is paving the way for therapeutic and investigational gene editing and modulation in a variety of organisms, including animals and humans. The ease of engineering and flexibility of CRISPR technology is projected to help solve numerous challenges associated with gene therapy. While much CRISPR research has focused on site-specific genome editing/disruption in vitro and in vivo, only a fraction of studies has focused on application of catalytically inactive Cas9 proteins for transcriptional modulation. For many clinical applications, transient transcriptional repression of a gene can provide a safer alternative to permanent gene disruption, which may alter germline DNA or create unintended genome mutations. In addition, most CRISPR-based studies have focused on modulating gRNA expression from ubiquitously active promoters. Efforts to improve regulatory control over CRISPR, such Spatiotemporally controlled CRISPR, have been limited. We recently combined logic-based design principles of synthetic biology with the function of the Cas9/CRISPR system to create CRISPR modulator circuits. Our CRISPR logic gene circuits carry internal regulatory controls that modulate Cas9 and gRNA expression or function after computation of two or more inputs. We propose to utilize these novel genetic circuits to develop safer, controllable CRISPR-based gene therapies to be tested in vitro and in vivo in liver. Effective delivery, safety, and control are all critical to the ultimate success of CRISPR in human. In this proposal, we have chosen to focus on safety and control. The approaches that we propose are designed with AAV?s payload limitation in mind. In the proposed project we will 1) Develop and validate spatiotemporal control over CRISPR gRNA through modulation by RNA Polymerase type II promoters.; 2) Establish Temporal control over CRISPR-based gene therapy for safer gene therapy approaches; 3) Establish a platform for CRISPR-mediated transcriptional interrogation of endogenous genes in response to injury in liver. We hypothesize that CRISPR toolset we develop and validate in this proposal can function reliably for controllable gene therapies. It will pave the way to more effective, safer gene and cell therapies for a variety of acquired and inherited diseases. ! !